Functional Genomics - Michael J. Brownstein , Ebooki - biologiczne [eng]

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Methods in Molecular Biology
TM
VOLUME 224
Functional
Methods and Protocols
Edited by
Michael J. Brownstein
Arkady B. Khodursky
Methods in Molecular Biology
TM
Functional
Genomics
Genomics
Methods and Protocols
Edited by
Michael J. Brownstein
Arkady B. Khodursky
1.
Fabrication of cDNA Microarrays
Xiang, Charlie C.; Brownstein, Michael J.
pp. 01-08
2.
Nylon cDNA Expression Arrays
Jokhadze, George; Chen, Stephen; Granger, Claire; Chenchik, Alex
pp. 09-30
3.
Plastic Microarrays: A Novel Array Support Combining the Benefi ts of Macro-
and Microarrays
Munishkin, Alexander; Faulstich, Konrad; Aivazachvili, Vissarion; Granger,
Claire; Chenchik, Alex
pp. 31-54
4.
Preparing Fluorescent Probes for Microarray Studies
Xiang, Charlie C.; Brownstein, Michael J.
pp. 55-60
5.
Escherichia coli Spotted Double-Strand DNA Microarrays: RNA Extraction,
Labeling, Hybridization, Quality Control, and Data Management
Khodursky, Arkady B.; Bernstein, Jonathan A.; Peter, Brian J.; Rhodius, Virgil;
Wendisch, Volker F.; Zimmer, Daniel P.
pp. 61-78
6.
Isolation of Polysomal RNA for Microarray Analysis
Arava, Yoav
pp. 79-88
7.
Parallel Analysis of Gene Copy Number and Expression Using cDNA
Microarrays
Pollack, Jonathan R.
pp. 89-98
8.
Genome-wide Mapping of Protein-DNA Interactions by Chromatin
Immunoprecipitation and DNA Microarray Hybridization
Lieb, Jason D.
pp. 99-110
9.
Statistical Issues in cDNA Microarray Data Analysis
Smyth, Gordon K.; Yang, Yee Hwa; Speed, Terry
pp. 111-136
10.
Experimental Design to Make the Most of Microarray Studies
Kerr, M. Kathleen
pp. 137-148
11.
Statistical Methods for Identifying Differentially Expressed Genes in DNA
Microarrays
Storey, John D.; Tibshirani, Robert
pp. 149-158
12.
Detecting Stable Clusters Using Principal Component Analysis
Ben-Hur, Asa; Guyon, Isabelle
pp. 159-182
13.
Clustering in Life Sciences
Zhao, Ying; Karypis, George
pp. 183-218
14.
A Primer on the Visualization of Microarray Data
Fawcett, Paul
pp. 219-234
15.
Microarray Databases: Storage and Retrieval of Microarray Data
Sherlock, Gavin; Ball, Catherine A.
pp. 235-248
Fabrication of cDNA Microarrays 1
1
Fabrication of cDNA Microarrays
Charlie C. Xiang and Michael J. Brownstein
1. Introduction
DNA microarray technology has been used successfully to detect the
expression of many thousands of genes, to detect DNA polymorphisms, and
to map genomic DNA clones
(1–4)
. It permits quantitative analysis of RNAs
transcribed from both known and unknown genes and allows one to compare
gene expression patterns in normal and pathological cells and tissues
(5
,
6)
.
DNA microarrays are created using a robot to spot cDNA or oligonucleotide
samples on a solid substrate, usually a glass microscope slide, at high densities.
The sizes of spots printed in different laboratories range from 75 to 150 µm
in diameter. The spacing between spots on an array is usually 100–200 µm.
Microarrays with as many as 50,000 spots can be easily fabricated on standard
25 mm × 75 mm glass microscope slides.
Two types of spotted DNA microarrays are in common use: cDNA and
synthetic oligonucleotide arrays
(7
,
8)
. The surface onto which the DNA is
spotted is critically important. The ideal surface immobilizes the target DNAs,
and is compatible with stringent probe hybridization and wash conditions
(9)
.
Glass has many advantages as such a support. DNA can be covalently attached
to treated glass surfaces, and glass is durable enough to tolerate exposure
to elevated temperatures and high-ionic-strength solutions. In addition, it is
nonporous, so hybridization volumes can be kept to a minimum, enhancing the
kinetics of annealing probes to targets. Finally, glass allows probes labeled with
two or more fl uors to be used, unlike nylon membranes, which are typically
probed with one radiolabeled probe at a time.
From:
Methods in Molecular Biology: vol. 224: Functional Genomics: Methods and Protocols
Edited by: M. J. Brownstein and A. Khodursky © Humana Press Inc., Totowa, NJ
1
 2 Xiang and Brownstein
2. Materials
1. Multiscreen fi ltration plates (Millipore, Bedford, MA).
2. Qiagen QIAprep 96 Turbo Miniprep kit (Qiagen, Valencia, CA).
3. dATP, dGTP, dCTP, and dTTP (Amersham Pharmacia, Piscataway, NJ).
4. M13F and M13R primers (Operon, Alameda, CA).
5.
Ta q
DNA polymerase and buffer (Invitrogen, Carlsbad, CA).
6. PCR CyclePlate (Robbins, Sunnyvale, CA).
7. CycleSeal polymerase chain reaction (PCR) plate sealer (Robbins).
8. Gold Seal microscope slides (Becton Dickinson, Franklin, NJ).
9. 384-well plates (Genetix, Boston, MA).
10. Succinic anhydride (Sigma, St. Louis, MO) in 325 mL of 1-methy-2-pyrrolidinone
(Sigma).
3. Methods
3.1. Selection and Preparation of cDNA Clones
3.1.1. Selection of Clones
Microarrays are usually made with DNA fragments that have been amplifi ed
by PCR from plasmid samples or directly from chromosomal DNA. The
sizes of the PCR products on our arrays range from 0.5 to 2 kb. They attach
well to the glass surface. The amount of DNA deposited per spot depends on
the pins chosen for printing, but elements with 250 pg to 1 ng of DNA (up to
9 × 10
8
molecules) give ample signals.
Many of the cDNA clones that have been arrayed by laboratories in the
public domain have come from the Integrated Molecular Analysis of Genomes
and Expression (IMAGE) Consortium set. Five million human IMAGE clones
have been collected and are available from Invitrogen/Research Genetics
(www.resgen.com/products/IMAGEClones.php3). Sequence-verifi ed cDNA
clones from humans, mice, and rats are also available from Invitrogen/Research
Genetics.
cDNA clones can also be obtained from other sources. The 15,000 National
Institute of Aging (NIA) mouse cDNA set has been distributed to many aca-
cDNA collections include the Brain Molecular Anatomy Project (BMAP)
clone sets. In preparing our arrays, we have used the NIA and BMAP collec-
tions and are in the process of sequencing the 5′ ends of the 41,000 clones in
the combined set in collaboration with scientists at the Korea Research Institute
of Bioscience and Biotechnology. Note that most cDNA collections suffer from
some gridding errors and well-to-well cross contamination.
 Fabrication of cDNA Microarrays 3
3.1.2. Preparation of Clones
Preparing DNA for spotting involves making plasmid minipreps, amplifying
their inserts, and cleaning up the PCR products. Most IMAGE clones are in
standard cloning vectors, and the inserts can be amplifi ed with modifi ed M13
primers. The sequences of the forward (M13F) and reverse (M13R) primers
used are 5′-GTTGTAAAACGACGGCCAGTG-3′ and 5′-CACACAGGAAA
CAGCTATG-3′, respectively. A variety of methods are available for purifying
cDNA samples. We use QIAprep 96 Turbo Miniprep kits and a Qiagen
BioRobot 8000 (Qiagen) for plasmid isolations but cheaper, semiautomated
techniques can be used as well. We PCR DNAs with a Tetrad MultiCycler
(MJ Research, Incline Village, NV) and purify the products with Multiscreen
fi ltration plates (Millipore).
3.1.3. Purifi cation of Plasmid
1. Culture the bacterial clones overnight in 1.3 mL of Luria–Bertani (LB) medium
containing 100 µg/mL of carbenicillin at 37°C, shaking them at 300 rpm in
96-well fl at-bottomed blocks.
2. Harvest the bacteria by centrifuging the blocks for 5 min at 1500
g
in an Eppendorf
centrifuge 5810R (Eppendorf, Westbury, NY). Remove the LB by inverting the
block. The cell pellets can be stored at –20°C.
3. Prepare cDNA using the BioRobot 8000, or follow the Qiagen QIAprep 96 Turbo
Miniprep kit protocol for manual extraction.
4. Elute the DNA with 100 µL of Buffer EB (10 m
M
Tris-HCl, pH 8.5) included in
the QIAprep 96 Turbo Miniprep kit. The plasmid DNA yield should be 5–10 µg
per prep.
3.1.4. PCR Amplifi cation
1. Dilute the plasmid solution 1
10 with 1X TE (10 m
M
Tris-HCl, pH 8.0, 1 m
M
EDTA).
2. For each 96-well plate to be amplifi ed, prepare a PCR reaction mixture containing
the following ingredients: 1000 µL of 10X PCR buffer (Invitrogen), 20 µL each
of dATP, dGTP, dCTP, and dTTP (100 m
M
each; Amersham Pharmacia), 5 µL
each of M13F and M13R (1 m
M
each; Operon), 100 µL of
Ta q
DNA polymerase
(5 U/µL; Invitrogen), and 8800 µL of ddH
2
O.
3. Add 100 µL of PCR reaction mix to each well of a PCR CyclePlate (Robbins)
plus 5 µL of diluted plasmid template. Seal the wells with CycleSeal PCR plate
sealer (Robbins). (Prepare two plates for amplifi cation from each original source
plate to give a fi nal volume of 200 µL of each product.)
4. Use the following PCR conditions: 96°C for 2 min; 30 cycles at 94°C for 30 s,
55°C for 30 s, 72°C for 1 min 30 s; 72°C for 5 min; and cool to ambient
temperature.
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